[1]王芳,黄娟,董乐,等.菠萝AcPIP1:2基因的克隆、重组载体的构建[J].泉州师范学院学报,2020,(02):17-23.
 WANG Fang,HUANG Juan,DONG Le,et al.Cloning and Constructing Expression Vector of AcPIP1:2 Gene from Pineapple[J].,2020,(02):17-23.
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菠萝AcPIP1:2基因的克隆、重组载体的构建()
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《泉州师范学院学报》[ISSN:1006-6977/CN:61-1281/TN]

卷:
期数:
2020年02期
页码:
17-23
栏目:
化学·生命科学
出版日期:
2020-04-15

文章信息/Info

Title:
Cloning and Constructing Expression Vector of AcPIP1:2 Gene from Pineapple
文章编号:
1009-8224(2020)02-0017-07
作者:
王芳1黄娟1董乐1陈明贤2刘宝13邱吕萍1陈月钗1
1.泉州师范学院 海洋与食品学院,福建 泉州 362000; 2.泉州市农业科学研究所,福建 泉州 362212; 3.东北师范大学 分子表观遗传学教育部重点实验室,吉林 长春 130024
Author(s):
WANG Fang1HUANG Juan1DONG Le1CHEN Mingxian2LIU Bao13QIU Lüping1CHEN Yuechai1
1.School of Oceanology and Food Science,Quanzhou Normal University,Quanzhou Fujian 362000,China; 2.Quanzhou Agricultural Research Institute,Quanzhou Fujian 362212,China; 3.Key Laboratory of Molecular Epigenetics of the Ministry of Education,Northeast Normal University,Changchun Jilin 130024,China
关键词:
菠萝 质膜水通道蛋白 载体构建 原核表达 体外转录
Keywords:
pineapple plasma membrane aquaporin vector construction prokaryotic expression transcription in vitro
分类号:
Q786
文献标志码:
A
摘要:
构建菠萝(Ananas comosus(Linn.)Merr.)质膜水通道蛋白基因(AcPIP1:2)原核表达载体和体外转录载体,为制备AcPIP1:2抗体和含有Poly(A)+的AcPIP1:2转录子奠定基础.以菠萝果实为试验材料,提取总RNA并反转录成cDNA.以cDNA为模板,根据GenBank中公布的AcPIP1:2全长cDNA序列(登录号:XM_020229679.1)设计合成特异性引物进行PCR扩增,获得AcPIP1:2开放阅读框(ORF)序列.序列分析表明,获得的AcPIP1:2序列与GenBank中公布的序列一致,序列长度为867 bp.该序列经Hind III和Xho I双酶切后亚克隆至原核表达载体pET32a(+),酶切、测序表明,成功构建了重组载体pET32a(+)-AcPIP1:2.将pET32a(+)-AcPIP1:2转到大肠杆菌表达菌株BL21(DE3)中表达,结果表明,异丙基硫代半乳糖苷(IPTG)诱导产生分子量为50 ku的融合蛋白.该序列经Hind III和Xba I双酶切后亚克隆至载体pSP64 Poly(A)中,酶切、测序表明,成功构建了重组载体pSP64 Poly(A)-AcPIP1:2.
Abstract:
The study aims to construct prokaryotic expression vector and transcription vector in vitro of plasma membrane aquaporin protein gene(AcPIP1:2)from pineapple(Ananas comosus(Linn.)Merr.),laying a foundation for the preparation of AcPIP1:2 antibody and AcPIP1:2 transcriptor containing Poly(A)+.Total RNA was extracted from pineapple fruit and the cDNA was synthesized by reverse transcription.Using cDNA as template,AcPIP1:2 open reading frame(ORF)sequence was amplified by RT-PCR with specific primers designed and synthesized according to the full-length cDNA sequence of AcPIP1:2 published in GenBank(Accession number:XM_020229679.1).Sequence analysis showed that the obtained AcPIP1:2 sequence was identical to the original sequence and its sequence length was 867 bp. AcPIP1:2 was sub-cloned into the prokaryotic expression vector pET32a(+)after the digestion with the enzyme Hind III and Xho I.The recombinant vectors pET32a(+)-AcPIP1:2 was successfully constructed,which was identified by endonuclease digestion and sequence analysis.pET32a(+)-AcPIP1:2 was transferred into the Escherichia coli expression strain BL21(DE3)for expression,and the results showed that IPTG induced the fusion protein with a molecular weight of 50 ku.AcPIP1:2 was sub-cloned into vector pSP64 Poly(A)after the digestion with the enzyme Hind III and Xba I.The recombinant vectors pET32a(+)-AcPIP1:2 was successfully constructed, which was identified by endonuclease digestion and sequence analysis.

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备注/Memo

备注/Memo:
收稿日期:2019-09-04
作者简介:王芳(1969-),女,蒙古族,辽宁瓦房店人,教授,博士,硕士生导师,主要从事植物分子生物学研究.
基金项目:泉州师范学院国家级和各部委项目预研基金(2016YYKJ17)
更新日期/Last Update: 2020-04-15